Nucleic acid encoding M. tuberculosis algu protein

ABSTRACT

The invention relates to  Mycobacterium tuberculosis  RNA polymerase algU sigma subunit protein, DNA encoding, and methods of detecting inhibitors of the RNA polymerase.

This application claims priority pursuant to 35 U.S.C. §119 fromProvisional Application Ser. No. 60/035,391 filed Jan. 16, 1997, theentire disclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Mycobacteria are gram-positive bacilli, nonmotile rod-shaped organismsthat do not form spores. The composition of the cell wall includes avery high concentration of lipids complexed to a variety of peptides andpolysaccharides. The unusual structure of the cell wall distinguishesmycobacteria from most other bacteria and is detectable by itsresistance to acid-alcohol staining.

The disease caused by M. tuberculosis is a progressive, deadly illnessthat tends to develop slowly and follows a chronic course (Plorde,1994). It is presently estimated that one-third of the world'spopulation is infected with M. tuberculosis, 30 million of whom haveactive disease (Plorde, 1994). An additional 8 million people developthe disease annually (Plorde, 1994). Most infections are caused byinhalation of droplet nuclei carrying the mycobacterium. A single coughcan generate 3000 infected droplet nuclei and even 10 bacilli may besufficient to cause a pulmonary infection. In addition to the primaryinfection, reactivation of the disease can occur in older people and inimmunocompromised patients.

When intracellular pathogens, such as Mycobacterium tuberculosis, areingested by macrophages the bacteria are under environmental stress. Thegenes required for survival following uptake by macrophages can provideinsight into mycobacterial pathogenesis, and provide novel targets fordeveloping antibacterial agents. The ability to adapt to theintracellular stress requires regulation of complex gene expression andthis regulation may be mediated in part by one or more alternative sigmafactors. Therefore stress response alternative sigma factors (sigEfamily) from M. tuberculosis are potential novel targets forantibacterial therapeutics.

Extracellular environmental stress can significantly affect the survivalof the bacteria. As part of the adaptive response by the bacteria thealternative sigma factors play a critical role in coordinate regulationof gene expression. For example, survival following extreme temperaturein Escherichia coli is regulated by a family of alternative sigmafactors known as the sigE family (Keiichiro et al., Raina et al.,Rouviere et al.). Alginate production in Pseudomonus aeruginosa is alsoregulated by the sigE family member known as the algU gene (Deretic etal.). Respiratory infections with mucoid P. aeruginosa in cysticfibrosis (CF) patients are the major cause of mortality. Althoughinitial colonizing strains are nonmucoid, the bacteria are converted tomucoid P. aeruginosa in the CF lung. This conversion to mucoidy isregulated by the alternative sigma factor algU (Martin et al.).

Sigma (σ) factors are positive regulators of general transcriptioninitiation that enhance transcriptional specificity. The basic unit ofthe eubacterial transcription apparatus is the DNA-dependent RNApolymerase holoenzyme, a complex consisting of five protein subunits:two copies of the α subunit and one copy each of the β, β′, and σsubunits. The α, β and β′ subunits are invariant in a given bacterialspecies and together form core RNA polymerase. Open promoter complexesform only when holoenzyme is bound at a promoter (Gross et al., 1992).When the newly synthesized RNA chain is 8-9 nucleotides long, σ factordissociates from the complex and the elongation process is begun (vonHippel, et al., 1992). After transcription is terminated, σ factorrebinds core polymerase, creating holoenzyme for another round ofinitiation (von Hippel, et al., 1992). This series of biochemicalactivities has been termed “the transcription cycle”.

Rifampicin, a highly specific inhibitor of mycobacterium/RNA polymerase,is one of the primary drugs of choice for treatment of tuberculosis.Combination treatment with isoniazid is typical if there is no risk ofdeveloping multi-drug resistance. Prolonged treatment regimens arenecessary and can take up to nine months. Failure to complete theprolonged treatment course is one of the contributing factors in thedevelopment of resistant bacterial strains. Rifabutin is an effectiveanalog of rifampicin, but 70% of rifampicin-resistant strains are alsorifabutin-resistant.

Although RNA polymerase is a well-validated target foranti-mycobacterial therapy, discovery of inhibitors of M. tuberculosisRNA polymerase is hampered by a lack of information concerningcomponents of the M. tuberculosis transcriptional apparatus,difficulties in obtaining sufficient yields of active enzymes forbiochemical studies, and technical and biosafety concerns surroundingthe handling of live cultures of M. tuberculosis. Establishment of an invitro transcription system employing purified and reconstituted RNApolymerase would greatly advance efforts to identify new therapeuticagents active against tuberculosis. It is very possible that moleculesthat inhibit a functions may not affect eukaryotic generaltranscription. Thus, σ factors are a reasonable target for developmentof transcriptional inhibitors. Therefore, molecules that inhibit σfactor function may be used as general transcriptional inhibitors andantibacterial therapeutics.

Accordingly, there is a need in the art for compositions and methodsutilizing cloned genes and purified proteins derived from M.tuberculosis RNA polymerase.

SUMMARY OF THE INVENTION

The present invention is based on the isolation and characterization ofDNA encoding the σ subunit of RNA polymerase derived from the algU genefrom M. tuberculosis. In one aspect, the invention provides a purified,isolated nucleic acid having the sequence shown in FIG. 3 SEQ ID NO:1.The invention also encompasses sequence-conservative andfunction-conservative variants of this sequence. The invention alsoprovides vectors comprising these sequences, and cells comprising thevectors.

In another aspect, the present invention provides a purified, isolatedpolypeptide encoded by the nucleic acid sequence shown in FIG. 3, aswell as function-conservative variants thereof.

In yet another aspect, the invention provides in vitro methods forhigh-throughput screening to detect inhibitors of M. tuberculosis RNApolymerase. The methods are carried out by the steps of:

a) providing a mixture comprising

(i) purified M. tuberculosis RNA polymerase containing the algU σ factorand

(ii) a DNA template encoding a promoter sequence that is recognized byM. tuberculosis RNA polymerase containing the algU subunit;

b) incubating the mixture in the presence of test compounds to form testsamples, and in the absence of test compounds to form control samples,under conditions that result in RNA synthesis in the control samples;

c) measuring RNA synthesis in the test and control samples; and

d) comparing the RNA synthesis detected in step (c) between the test andcontrol samples. According to the invention, an RNA polymerase inhibitoris a test compound that causes a reduction in RNA synthesis measured inthe test sample relative to RNA synthesis measured in the controlsample.

In yet another aspect, the invention provides in vivo methods forhigh-throughput screening to detect inhibitors of M. tuberculosis RNApolymerase. The methods are carried out by the steps of:

a) providing a non-mycobacterial bacterial strain, preferably E. coli,that

(i) has been transformed with a DNA template encoding a promotersequence that is recognized by M. tuberculosis RNA polymerase containingthe algU subunit, and

(ii) expresses enzymatically active M. tuberculosis RNA polymerase(e.g., α, β, β′ plus the algU σ subunit disclosed herein);

b) incubating the bacterial strain of (a) in the presence of testcompounds to form test samples, and in the absence of test compounds toform control samples;

c) measuring RNA synthesis in the test and control samples; and

d) comparing the RNA synthesis detected in step (c) between the test andcontrol samples. According to the invention, an RNA polymerase inhibitoris a test compound that causes a reduction in RNA synthesis measured inthe test sample relative to RNA synthesis measured in the controlsample.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in light of the present specificationand appended claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1. PCR amplification of M. tuberculosis H37Rv genomic DNA. lane M:DNA marker (123 bp, Gibco-BRL), lane 1: primer P1 only, lane 2: primerP2 only, lane 2: primer P2 only and lane 3: primer P1 and P2. Theamplified DNA fragment (arrow in FIG. 1) was gel purified and subclonedinto pCRScript (Stratagene) plasmid.

FIG. 2A. Southern blot analysis of M. tuberculosis H37Rv DNA and cosmidclones. A. M. tuberculosis H37Rv genomic DNA were digested withrestriction enzymes: BamH I (lane 1), Pst I (lane 2), Pvu II (lane 3),Sma I (lane 4) and Xmn I (lane5) and analyzed by Southern hybridizationusing the PCR amplified DNA fragment as a probe. Sizes of DNA markers(³⁵S-DNA Marker, Amersham) are indicated in kb. 2B. Two differentpositive clones (designated 2D11 and 4D11) isolated from an M.tuberculosis cosmid library were digested with BamH I (lane 1 and 4),Pvu II (lane 2 and 5) and Sma I (lane 3 and 6) and hybridized with thePCR-generated sigma gene as a probe.

FIG. 3. Nucleotide and deduced amino acid sequences of the M.tuberculosis H37Rv algU gene SEQ ID NO:1 and SEQ ID NO:2, respectively.

FIG. 4. Alignment of the inferred amino acid sequence of the M.tuberculosis (Mt) H37Rv algU gene with sequences of extracellularfunction family of sigma subunits from other bacteria (Streptomycescoelicolor SEQ ID NO:3, Pseudomonas aeruginosa SEQ ID NO:4, Escherechiacoli SEQ ID NO:5 and Hemophilus influenzae SEQ ID NO:6) Shadingindicates identical amino acid residues. Amino acid sequence alignmentswere performed using MegAlign (DNAStar).

DETAILED DISCUSSION OF THE INVENTION

All patents, patent applications and literature references cited hereinare hereby incorporated in their entirety. In the case ofinconsistencies, the present disclosure will prevail.

The present invention is based on the isolation of a fragment of the M.tuberculosis algU gene, encoding an alternative σ subunit of RNApolymerase. As described in Example 1 below. PCR amplification of M.tuberculosis genomic DNA with primers based on the M. leprae algU DNAsequence generated an expected size of DNA (180 base pairs) (FIG. 1).The PCR amplified DNA had >90% identity to the M. leprae gene. Southernblot analysis demonstrated the presence of a single copy of this gene inM. tuberculosis (FIG. 2A). The amplified DNA was utilized as ahybridization probe to recover the entire algU gene from a cosmidlibrary of genomic DNA from virulent M. tuberculosis strain H37RV.Nucleotide sequencing indicated that the 675 bp M. tuberculosis algUopen reading frame (ORF) encodes a protein of 24.3 kDa (225 amino acids)which shows significant structural similarity to the σ subunits ofdiverse bacterial species with greatest identity to the stress relatedextracellular function family of σ subunits of Streptomyces coelicolor,Pseudomonas aeruginosa, Escherochia coli and Hemophius influenzae. Thesigma factors from S. coelicolor SEQ ID NO:3 and P. aeruginosa , SEQ IDNO4, E. coli and H. influenzae SEQ ID NO:6 are 24%, 20%, 21% and 16%identical to the M. tuberculosis sequence SEQ ID NO:5 respectively (FIG.4).

The P. aeruginosa algU gene is part of a large operon that containsgenes for anti-sigma factors (mucA and mucB) and a protease (mucD)(Schurr et al.). Further nucletodide sequencing and availability of anintegrated map of the genome of M. tuberculosis H37Rv (Philipp et al.,1996) is expected to clarify the structural organization and position ofthe algU locus of M. tuberculosis.

In practicing the present invention, many techniques in molecularbiology, microbiology, recombinant DNA, and protein biochemistry such asthese explained fully in, for example, Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A PracticalApproach, Volumes I and II, 1985 (D.N. Glover ed.); OligonucleotideSynthesis, 1984, (M. L. Gait ed.); Transcription and Translation, 1984(Hames and Higgins eds.); A Practical Guide to Molecular Cloning; theseries, Methods in Enzymology (Academic Press, Inc.); and ProteinPurification: Principles and Practice, Second Edition (Springer-Verlag,N.Y.), may be used.

The present invention encompasses nucleic acid sequences encoding thealgU gene of M. tuberculosis, enzymatically active fragments derivedtherefrom, and related sequences. As used herein, a nucleic acid that is“derived from” a sequence refers to a nucleic acid sequence thatcorresponds to a region of the sequence, sequences that are homologousor complementary to the sequence, and “sequence-conservative variants”and “function-conservative variants”. Sequence-conservative variants arethose in which a change of one or more nucleotides in a given codonposition results in no alteration in the amino acid encoded at thatposition. Function-conservative variants are those in which a givenamino acid residue in the algU subunit has been changed without alteringthe overall conformation and function of the polypeptide, including, butnot limited to, replacement of an amino acid with one having similarphysico-chemical properties (such as, for example, acidic, basic,hydrophobic, and the like). Fragments of the algU subunit that retainenzymatic activity can be identified according to the methods describedherein, e.g., expression in E. coli followed by enzymatic assay of thecell extract.

The nucleic acids of the present invention include purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides.This includes single- and double-stranded molecules, i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases. The nucleic acids maybe isolated directly from cells. Alternatively, PCR can be used toproduce the nucleic acids of the invention, using either chemicallysynthesized strands or genomic material as templates. Primers used forPCR can be synthesized using the sequence information provided hereinand can further be designed to introduce appropriate new restrictionsites, if desirable, to facilitate incorporation into a given vector forrecombinant expression.

The nucleic acids of the present invention may be flanked by natural M.tuberculosis regulatory sequences, or may be associated withheterologous sequences, including promoters, enhancers, responseelements, signal sequences, polyadenylation sequences, introns, 5′- and3′-noncoding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Nucleic acids may containone or more additional covalently linked moieties, such as, for example,proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The nucleic acid may be derivatized by formationof a methyl or ethyl phosphotriester or an alkyl phosphoramidatelinkage. Furthermore, the nucleic acid sequences of the presentinvention may also be modified with a label capable of providing adetectable signal, either directly or indirectly. Exemplary labelsinclude radioisotopes, fluorescent molecules, biotin, and the like.

The invention also provides nucleic acid vectors comprising thedisclosed algU subunit sequences or derivatives or fragments thereof. Alarge number of vectors, including plasmid and fungal vectors, have beendescribed for replication and/or expression in a variety of eukaryoticand prokaryotic hosts. Non-limiting examples include pKK plasmids(Clontech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.),or pRSET or pREP (Invitrogen, San Diego, Calif.), and many appropriatehost cells, using methods disclosed or cited herein or otherwise knownto those skilled in the relevant art. Recombinant cloning vectors willoften include one or more replication systems for cloning or expression,one or more markers for selection in the host, e.g. antibioticresistance, and one or more expression cassettes. Suitable host cellsmay be transformed/transfected/infected as appropriate by any suitablemethod including electroporation, CaCl₂ mediated DNA uptake, fungalinfection, microinjection, microprojectile, or other establishedmethods.

Appropriate host cells include bacteria, archebacteria, fungi,especially yeast, and plant and animal cells, especially mammaliancells. Of particular interest are E. coli, B. subtilis, Saccharomycescerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombe, SF9cells, C129 cells, 293 cells, Neurospora, and CHO cells, COS cells, HeLacells, and immortalized mammalian myeloid and lymphoid cell lines.Preferred replication systems include M13, ColE1, SV40, baculovirus,lambda, adenovirus, and the like. A large number of transcriptioninitiation and termination regulatory regions have been isolated andshown to be effective in the transcription and translation ofheterologous proteins in the various hosts. Examples of these regions,methods of isolation, manner of manipulation, etc. are known in the art.Under appropriate expression conditions, host cells can be used as asource of recombinantly produced Mycobacterial-derived peptides andpolypeptides.

Advantageously, vectors may also include a transcription regulatoryelement (i.e., a promoter) operably linked to the algU subunit portion.The promoter may optionally contain operator portions and/or ribosomebinding sites. Non-limiting examples of bacterial promoters compatiblewith E. coli include: trc promoter, β-lactamase (penicillinase)promoter; lactose promoter; tryptophan (trp) promoter; arabinose BADoperon promoter; lambda-derived P1 promoter and N gene ribosome bindingsite; and the hybrid tac promoter derived from sequences of the trp andlac UV5 promoters. Non-limiting examples of yeast promoters include3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphatedehydrogenase (GAPDH) promoter, galactokinase (GALI) promoter,galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter.Suitable promoters for mammalian cells include without limitation viralpromoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus(RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammaliancells may also require terminator sequences and poly A additionsequences, and enhancer sequences which increase expression may also beincluded. Sequences which cause amplification of the gene may also bedesirable. Furthermore, sequences that facilitate secretion of therecombinant product from cells, including, but not limited to, bacteria,yeast, and animal cells, such as secretory signal sequences and/orprohormone pro region sequences, may also be included.

Nucleic acids encoding wild-type or variant subunit polypeptides mayalso be introduced into cells by recombination events. For example, sucha sequence can be introduced into a cell, and thereby effect homologousrecombination at the site of an endogenous gene or a sequence withsubstantial identity to the gene. Other recombination-based methods,such as non-homologous recombinations or deletion of endogenous genes byhomologous recombination, may also be used.

algU subunit-derived polypeptides according to the present invention,including function-conservative variants, may be isolated from wild-typeor mutant M. tuberculosis cells, or from heterologous organisms or cells(including, but not limited to, bacteria, fungi, insect, plant, andmammalian cells) into which a subunit-derived protein-coding sequencehas been introduced and expressed. Furthermore, the polypeptides may bepart of recombinant fusion proteins. Alternatively, polypeptides may bechemically synthesized by commercially available automated procedures,including, without limitation, exclusive solid phase synthesis, partialsolid phase methods, fragment condensation or classical solutionsynthesis.

“Purification ” of a σ subunit polypeptide refers to the isolation ofthe polypeptide in a form that allows its enzymatic activity to bemeasured without interference by other components of the cell in whichthe polypeptide is expressed. Methods for polypeptide purification arewell-known in the art, including, without limitation, preparativedisc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phaseHPLC, gel filtration, ion exchange and partition chromatography, andcountercurrent distribution. For some purposes, it is preferable toproduce the polypeptide in a recombinant system in which the proteincontains an additional sequence tag that facilitates purification, suchas, but not limited to, a polyhistidine sequence. The polypeptide canthen be purified from a crude lysate of the host cell by chromatographyon an appropriate solid-phase matrix. Alternatively, antibodies producedagainst the σ subunit or against peptides derived therefrom can be usedas purification reagents. Other purification methods are possible.

The isolated polypeptides may be modified by, for example,phosphorylation, sulfation, acylation, or other protein modifications.They may also be modified with a label capable of providing a detectablesignal, either directly or indirectly, including, but not limited to,radioisotopes and fluorescent compounds.

Screening Methods to Identify Anti-tuberculosis Agents

The methods and compositions of the present invention can be used toidentify compounds that inhibit the function of M. tuberculosis RNApolymerase and thus are useful as anti-tuberculosis agents. This isachieved by providing active recombinant algU subunit according to thepresent invention, in combination with other components of RNApolymerase, in a context in which the inhibitory effects of testcompounds can be measured.

In a preferred embodiment, recombinant M. tuberculosis RNA polymerasesubunits (α, β, β′ plus the σ subunit disclosed herein) are purified inmilligram quantities from E. coli cultures by affinity methods utilizinga hexahistidine tagged α and σ subunits. Enzymatically active holoenzymeis reconstituted using these components. The active polymerase is thenincubated in the presence of test compounds to form test mixtures, andin the absence of test compounds to form control mixtures. In vitrotranscription is then carried out using a DNA template containingappropriate promoter and reporter sequences. (See Example 3 below.)

In another embodiment, M. tuberculosis RNA polymerase subunits (α, β, β′plus the σ subunit disclosed herein) are co-expressed in E. coli oranother surrogate bacterial cell, in conjunction with an appropriatepromoter-reporter gene. The ability of test compounds to differentiallyinhibit M. tuberculosis RNA polymerase is then assessed.

M. tuberculosis promoters useful in practicing the invention includewithout limitation: hsp 60 promoter (Stover et al., 1991); cpn-60promoter (Kong et al., 1993); 85A antigen promoter (Kremer, 1995); PANpromoter (Murray et al., 1992); 16S RNA promoter (Ji et al., 1994); andasks promoter (Cirillo et al., 1994). Useful reporter genes includewithout limitation xy1E (Curcic et al., 1994); CAT (Das Gupta et al.,1993); luciferase (Cooksey et al., 1993); green fluorescent protein(Dhadayuthap et al., 1995); and lacZ (Silhavy et al., 1985).

It will be understood that the present invention encompasses M.tuberculosis RNA polymerases containing the algU σ factor disclosedherein, which is used in conjunction with particular promoters that arerecognized by RNA polymerase containing this σ factor. The inventionalso encompasses the identification of additional promoters that arerecognized by the particular σ subunit of the present invention. This isachieved by providing a library of random M. tuberculosis gene fragmentscloned upstream of an appropriate reporter gene (see above). The libraryis transformed into M. tuberculosis or M. smegmatis and reporter geneexpression is measured. Alternatively, the library is transformed intoanother bacterial cell, such as, e.g., E. coli, which expresses M.tuberculosis RNA polymerase core subunits as well as the σ subunit ofthe present invention and cognate promoters that drive reporter geneexpression. In yet another embodiment, expression of an M. tuberculosisσ factor confers new recognition properties on E. coli RNA polymeraseand permits isolation of promoters utilized specifically by a particularM. tuberculosis σ subunit.

Preferably, both in vitro and in vivo screening methods of the presentinvention are adapted to a high-throughput format, allowing amultiplicity of compounds to be tested in a single assay. Suchinhibitory compounds may be found in, for example, natural productlibraries, fermentation libraries (encompassing plants andmicroorganisms), combinatorial libraries, compound files, and syntheticcompound libraries. For example, synthetic compound libraries arecommercially available from Maybridge Chemical Co. (Trevillet, Cornwall,UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H),and Microsource (New Milford, Conn.). A rare chemical library isavailable from Aldrich Chemical Company, Inc. (Milwaukee, Wis.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available from, for example, PanLaboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readilyproducible. Additionally, natural and synthetically produced librariesand compounds are readily modified through conventional chemical,physical, and biochemical means (Blondelle et al., TibTech 14:60, 1996).preferably using automated equipment, to allow for the simultaneousscreening of a multiplicity of test compounds.

Useful anti-tuberculosis compounds are identified as those testcompounds that decrease tuberculosis-specific transcription. Once acompound has been identified by the methods of the present invention asan RNA polymerase inhibitor, in vivo and in vitro tests may be performedto further characterize the nature and mechanism of the inhibitoryactivity. For example, classical enzyme kinetic plots can be used todistinguish, e.g., competitive and non-competitive inhibitors.

Compounds identified as RNA polymerase inhibitors using the methods ofthe present invention may be modified to enhance potency, efficacy,uptake, stability, and suitability for use in pharmaceuticalformulations, etc. These modifications are achieved and tested usingmethods well-known in the art.

The present invention is further described in the following exampleswhich are intended to further describe the invention without limitingthe scope thereof.

EXAMPLE 1

In the present Example, the following Materials and Methods were used.

PCR amplification: Based on the M. leprae cosmid sequence (cosmidB-1620, Genbank accession #U-00015, position 36121-35942), a set ofprimers was designed and the sequence of these primers was:5′-ATGAACGAACTGCTCGAGATCTTGCCTGCC-3′ (P1) SEQ ID NO:7 and5′-TCACCCGCCGCGACGATCTCGGACGTCAAC-3′(P2) SEQ ID NO:8. Amplification wasperformed using 100 ng of M. tuberculosis H37Rv genomic DNA using aprogrammable thermal controller (PTC100, M J Research, Inc.). The PCRconditions were as follows: reaction volume 100 μl; pfu cloned DNApolymerase (Stratagene); 0.2 mM dNTPs (Boehringer-Mannheim); 100 ng ofprimer; one cycle of 94° C. for 1 minute, thirty cycle of 94° C. for oneminute, 50° C. for one minute and 72° C. for one minute.

Southern blot analysis: Restriction enzyme digests of M. tuberculosisH37Rv chromosomal DNAs were electrophoresed on 1% TAE-agarose gels andtransferred to nytran membranes (Schleicher and Schuell) using aPressure Blotter (Stratagene). Probe labeling was performed using therediprime DNA labelling system (Amersham) essentially as described bythe supplier. Hybridization was performed using 6×SSC, 5×Denhardtsolution, 0.5% SDS, 0.1 mg per ml Salmon Sperm DNA and 50% formamide.Washing was performed using 2×SSC, 0.5%SDS, at room temperature for 15min and 0.1×SSC, 0.5% SDS at 37° C. for 15 min.

Cosmid hybridizations: A transducing lysate of a cosmid library of M.tuberculosis H37Rv genomic DNA in vector pYA3060 was generously suppliedby Dr. J. Clark-Curtiss. Cosmid-bearing E. coli χ2819T (Jacobs et al,1986) colonies representing roughly five genomic equivalents wereindividually picked to wells of sterile 96-well microtiter dishes andpropagated at 30° C. in Luria broth containing ampicillin at 30 μg/mland thymidine at 50 μg/ml. Colonies were grown overnight at roomtemperature on the above media as nylon filter replicas of the library.Filters were processed for colony hybridization by standard methods andprobe hybridizations performed as described above. Cosmid DNAs werepurified using maxiprep columns (Qiagen).

DNA sequencing and analysis: Plasmid templates for nucleotide sequencingwere purified using maxiprep columns (Qiagen). PCR cycle sequencing (ABIPrizm) was carried out with an Applied Biosystems automated sequencer atthe Massachusetts General Hospital DNA Sequencing Core Facility,Department of Molecular Biology (Boston, Mass.).

(a) Cloning of M. tuberculosis algU Gene

A DNA fragment (180base pair) that contains the M. tuberculosis algUgene was identified by using PCR amplification of M. tuberculosis H37Rvgenomic DNA with primers that were derived from the M. leprae cosmidsequence. To determined whether the amplified DNA fragment contains thealgU gene, the 180 base pair DNA fragment was subcloned into a pCRScript(Stratagene) plasmid and nucleotide sequences were determined. Thededuced amino acid sequence of the PCR product showed significanthomology to the algU sequence from other bacteria (FIG. 4).

(b) Southern Blot Analysis and Isolation of the Full Length M.tuberculosis algU Gene

To see whether the cloned algU gene is a single copy of gene in M.tuberculosis Southern blot analysis was performed. The PCR cloned DNAfragment was used as a probe to analyzed the M. tuberculosis H37Rvgenomic DNA that was digested with endonucleases. The PCR cloned DNAprobe recognized a single band in each digested chromosomal DNA (FIG.2), and it was concluded that the algU gene is a single copy of gene inM. tuberculosis.

The full-length algU gene was obtained from a cosmid library of M.tuberculosis H37Rv genomic fragments (kindly provided by Dr. J.Clark-Curtiss) using the 180 bp as a probe. Screening of 552cosmid-bearing E. coli colonies (representing roughly 5 genomeequivalents) with the algU gene fragment yielded 5 positive clones. OnealgU-hybridizing cosmid clone, 4D11 was analyzed, and Southern blottingof 4D11 DNA digested with a panel of restriction enzymes confirmed thatthe no gross structural rearrangements of the algU gene had occurredduring cloning (FIG. 3) SEQ ID NO:1. The 1.1 kb BamH I, 1.2 kb PvuII and1 kb Sma I algU-hybridizing fragments of cosmid 4D11 were subcloned intovector pSKII+ prior to nucleotide sequencing.

(c) Sequence Analysis of the M. tuberculosis algU Gene

Nucleotide sequencing was performed on plasmid subclones shown in FIG.4. The sequence encodes a 675 bp ORF which has an overall G+Ccomposition of 63% (85% for bases occupying the codon third position).Assuming that the ATG at position 53-5 serves as the initiator codon,the ORF is expected to encode a protein of 225 amino acids. A strongmatch with the consensus sequence for an M. tuberculosis ribosomebinding site (CAGGTG), (Novick, 1996) is positioned just upstream of theputative ATG codon. Examination of more than 63 bp of nucleotidesequence upstream of the translation start site did not reveal regionsof exact identity with prokaryotic promoter sites. Among σ subunitsstudied in other bacterial species, the deduced amino acid sequence ofthe 225 residue M. tuberculosis protein displayed greatest similarity tothe stress related extracellular function family of sigma subunits ofStreptomyces coelicolor, Pseudomonas aeruginosa, Escherechia coli andHemophilus influenzae (FIG. 4) SEQ ID NOS:3,4,5, and 6, respectively.

EXAMPLE 2

Others have shown that overexpressed E. coli RNA polymerase subunits canbe reconstituted into an enzymatically active protein (Zalenskaya etal., 1990; Kashlev et al., 1993; Tang et al., 1995). The M. tuberculosisrpoA (Healy et al.), rpoB and rpoC genes (Miller et al., 1994) have beencloned and characterized. Using the overexpressed M. tuberculosis RNApolymerase subunits, the in vitro reconstitution assay to form theenzymatically active core enzyme will be performed. Holoenzyme thatcontains algU sigma subunit can be obtained and biochemical analysis ofgene regulation in M. tuberculosis will be studied. Transcriptioninhibitors that act against the holoenzyme that contains the stressrelated sigma factor will be identified.

EXAMPLE 3

High Throughput Screens for Inhibitors of M. tuberculosis RNA Polymeraseand σ Subunit

High-throughput screens for anti-tuberculosis agents may be performedusing either an in vitro or in vivo format. In either case, the abilityof test compounds to inhibit M. tuberculosis RNA polymerase-driventranscription of M. tuberculosis promoters is tested.

The algU sigma factor of the present invention regulates transcriptionof promoters characterized by the sigma promotor consensus sequence:GAACTT-(N16/17)-TCTgA-N(1-5)SEQ ID NO:9 (Deretic, et al., 1994;Erickson, et al., 1989; Lipnska, et al., 1988; Martin, et al., 1994;Scharr, et al., 1995). Therefore, this promoter is preferred for useherein.

a) In vitro screens:

The following procedure is used for cell-free high-throughput screening.A Tomtec Quadra 96-well pipetting station is used to add the reactioncomponents to polypropylene 96-well dishes. 5 μl aliquots of testcompounds dissolved in DMSO (or DMSO alone as a control) are added towells. This is followed by 20 μl of the RNA polymerase mixture, whichconsists of: 10 mM DTT, 200 mM KCl, 10 mM Mg⁺², 1.5 μM bovine serumalbumin, and 0.25 μg reconstituted RNA polymerase. After allowing thetest compound to interact with the RNA polymerase, 25 μl of the DNA/NTPmixture is added, containing: 1 μg template DNA (see above), 4 μM[α-³²P]-UTP, and 400 μM each CTP, ATP, and GTP.

After incubation for 30 min at 25° C., the reaction is stopped byaddition of 150 μl 10% trichloroacetic acid (TCA). After incubation atroom temperature for 60 min, the TCA-precipitated RNA is adsorbed ontodouble-thick glass fiber filtermats using a Tomtec cell harvester. Thewells of the microtiter plate and the filter are washed twice with 5%TCA and bound radioactivity is determined using a Wallac microbeta 1450scintillation counter.

Inhibitory activity due to the test compound is calculated according tothe formula:${\% \quad {inhibition}} = {\frac{\left( {{cpm}_{{positive}\quad {control}} - {cpm}_{sample}} \right)}{{cpm}_{{positive}\quad {control}}} \times 100}$

where cpm_(positive control) represents the average of the cpm in wellsthat received DMSO alone, and cpm_(sample) represents the cpm in thewell that received test compound. Compounds that cause at least 50%inhibition are scored as positive “hits” in this assay.

As an additional control, rifampicin is used at a concentration of 30nM, which results in a 50-75% inhibition of transcription in this assay.

b) In vivo screen:

M. tuberculosis RNA polymerase subunits (α, β, β′, and the σ subunitdisclosed herein) are expressed in E. coli under the control ofregulatable promoters by transforming E. coli with appropriate plasmids.If the σ subunit is expressed, a DNA sequence comprising the sigEpromoter described above is also introduced into the cells to serve as atemplate for M. tuberculosis-specific transcription.

In one embodiment, the sigE promoter sequence is linked to a DNAsequence encoding the xylE gene product, catechol 2, 3-dioxygenase(CDO). When expressed in the E. coli cell, CDO converts catechol to2-hydroxymuconic semialdehyde, which has a bright yellow color (havingan absorbance maximum at 375 nm) that is easily detected in whole cellsor in crude extracts. The substrate for this enzyme is a small aromaticmolecule that easily enters the bacterial cytoplasm and does notadversely affect cell viability.

In a high-throughput format, aliquots of bacterial cultures areincubated in the absence or presence of test compounds, and CDO activityis monitored by measuring absorbance at 375 nm following addition ofcatechol.

c) Specificity:

Compounds that score as positive in either the in vitro or in vivoassays described above are then tested for their effect on human RNApolymerase II. Those compounds which do not significantly inhibit humanRNA polymerase II will be further developed as potentialanti-tuberculosis agents.

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9 1 874 DNA Mycobacteria tuberculosis 1 gtaacgttgg agatatcgcc gtcgatgacaatgcaagggg aacgtctcga cgctgtggtt 60 gcggaggccg tggcaggaga ccggaacgcgcttcgggagg tgctggagac catccgcccg 120 atcgtcgtgc gatattgccg agcgcgagtcggcacggtcg agcggagcgg cctgtcagca 180 gatgacgtgg cacaggaggt gtgcttggccaccataacgg cgctgccgcg ctatcgggac 240 cgcggccggc cattcctggc gtttctgtacggcatcgcgg cgcacaaggt tgccgacgcc 300 catcgggcag ccggccgtga ccgggcctatcccgccgaaa cgcttcctga gcgctggtca 360 gccgacgccg gcccggagca gatggccatcgaggccgatt cggtcacccg gatgaacgaa 420 ttgcttgaga tcttgccggc caagcaacgcgagatcctca ttctgcgtgt tgtcgtcggc 480 ctgtccgcgg aagagaccgc cgccgccgtcggcagcacca cgggggcggt ccgggtggcc 540 caacaccgtg cacttcagcg gctgaaggacgaaattgttg cggcaggtga ctatgcgtga 600 atttggtaat ccccttggcg atcggccgccattggatgag ctggcccgca ccgatctgct 660 gctcgacgca ctcgccgaac gggaggaggttgacttcgcg gatcctcgcg atgacgcgtt 720 ggccgccctg ctcggacagt ggcgcgacgacttgaggtgg ccgccggcca gtgccctggt 780 ttcacaggac gaggccgtcg ccgcgttgcgcgccggggta gcgcaacggc gacgggctcg 840 tcgcagcctg gcggccgtcg ggtcggtggccgcg 874 2 224 PRT Mycobacteria tuberculosis 2 Met Leu Ala Tyr Arg LeuLys Arg Gly Trp Ala Val Met Val Asp Pro 1 5 10 15 Gly Val Ser Pro GlyCys Val Arg Phe Val Thr Leu Glu Ile Ser Pro 20 25 30 Ser Met Thr Met GlnGly Glu Arg Leu Asp Ala Val Val Ala Glu Ala 35 40 45 Val Ala Gly Asp ArgAsn Ala Leu Arg Glu Val Leu Glu Thr Ile Arg 50 55 60 Pro Ile Val Val ArgTyr Cys Arg Ala Arg Val Gly Thr Val Glu Arg 65 70 75 80 Ser Gly Leu SerAla Asp Asp Val Ala Gln Glu Val Cys Leu Ala Thr 85 90 95 Ile Thr Ala LeuPro Arg Tyr Arg Asp Arg Gly Arg Pro Phe Leu Ala 100 105 110 Phe Leu TyrGly Ile Ala Ala His Lys Val Ala Asp Ala His Arg Ala 115 120 125 Ala GlyArg Asp Arg Ala Tyr Pro Ala Glu Thr Leu Pro Glu Arg Trp 130 135 140 SerAla Asp Ala Gly Pro Glu Gln Met Ala Ile Glu Ala Asp Ser Val 145 150 155160 Thr Arg Met Asn Glu Leu Leu Glu Ile Leu Pro Ala Lys Gln Arg Glu 165170 175 Ile Leu Ile Leu Arg Val Val Val Gly Leu Ser Ala Glu Glu Thr Ala180 185 190 Ala Ala Val Gly Ser Thr Thr Gly Ala Val Arg Val Ala Gln HisArg 195 200 205 Ala Leu Gln Arg Leu Lys Asp Glu Ile Val Ala Ala Gly AspTyr Ala 210 215 220 3 177 PRT Streptomyces coelicolor 3 Met Gly Glu ValLeu Glu Phe Glu Glu Tyr Val Arg Thr Arg Gln Asp 1 5 10 15 Ala Leu LeuArg Ser Ala Arg Arg Leu Val Pro Asp Pro Val Asp Ala 20 25 30 Gln Asp LeuLeu Gln Thr Ala Leu Ala Arg Thr Tyr Gly Arg Trp Glu 35 40 45 Thr Ile GluAsp Lys Arg Leu Ala Asp Ala Tyr Leu Arg Arg Val Met 50 55 60 Ile Asn ThrArg Thr Glu Trp Trp Arg Ala Arg Lys Leu Glu Glu Val 65 70 75 80 Pro ThrGlu Gln Leu Pro Glu Ser Pro Met Asp Asp Ala Thr Glu Gln 85 90 95 His AlaAsp Arg Ala Leu Leu Met Asp Val Leu Lys Val Leu Ala Pro 100 105 110 LysGln Arg Ser Val Val Val Leu Arg His Trp Glu Gln Met Ser Thr 115 120 125Glu Glu Thr Ala Ala Ala Leu Gly Met Ser Ala Gly Thr Val Lys Ser 130 135140 Thr Leu His Arg Ala Leu Ala Arg Leu Arg Glu Glu Leu Val Ala Arg 145150 155 160 Asp Leu Asp Ala Arg Ala Leu Glu Arg Glu Glu Arg Glu Arg CysAla 165 170 175 Ala 4 193 PRT Pseudomonas aeruginosa 4 Met Leu Thr GlnGlu Gln Asp Gln Gln Leu Val Glu Arg Val Gln Arg 1 5 10 15 Gly Asp LysArg Ala Phe Asp Leu Leu Val Leu Lys Tyr Gln His Lys 20 25 30 Ile Leu GlyLeu Ile Val Arg Phe Val His Asp Ala Gln Glu Ala Gln 35 40 45 Asp Val AlaGln Glu Ala Phe Ile Lys Ala Tyr Arg Ala Leu Gly Asn 50 55 60 Phe Arg GlyAsp Ser Ala Phe Tyr Thr Trp Leu Tyr Arg Ile Ala Ile 65 70 75 80 Asn ThrAla Lys Asn His Leu Val Ala Arg Gly Arg Arg Pro Pro Asp 85 90 95 Ser AspVal Thr Ala Glu Asp Ala Glu Phe Phe Glu Gly Asp His Ala 100 105 110 LeuLys Asp Ile Glu Ser Pro Glu Arg Ala Met Leu Arg Asp Glu Ile 115 120 125Glu Ala Thr Val His Gln Thr Ile Gln Gln Leu Pro Glu Asp Leu Arg 130 135140 Thr Ala Leu Thr Leu Arg Glu Phe Glu Gly Leu Ser Tyr Glu Asp Ile 145150 155 160 Ala Thr Val Met Gln Cys Pro Val Gly Thr Val Arg Ser Arg IlePhe 165 170 175 Arg Ala Arg Glu Ala Ile Asp Lys Ala Leu Gln Pro Leu LeuArg Glu 180 185 190 Ala 5 191 PRT Escherichia coli 5 Met Ser Glu Gln LeuThr Asp Gln Val Leu Val Glu Arg Val Gln Lys 1 5 10 15 Gly Asp Gln LysAla Phe Asn Leu Leu Val Val Arg Tyr Gln His Lys 20 25 30 Val Ala Ser LeuVal Ser Arg Tyr Val Pro Ser Gly Asp Val Pro Asp 35 40 45 Val Val Gln GluAla Phe Ile Lys Ala Tyr Arg Ala Leu Asp Ser Phe 50 55 60 Arg Gly Asp SerAla Phe Tyr Thr Trp Leu Tyr Arg Ile Ala Val Asn 65 70 75 80 Thr Ala LysAsn Tyr Leu Val Ala Gln Gly Arg Arg Pro Pro Ser Ser 85 90 95 Asp Val AspAla Ile Glu Ala Glu Asn Phe Glu Ser Gly Gly Ala Leu 100 105 110 Lys GluIle Ser Asn Pro Glu Asn Leu Met Leu Ser Glu Glu Leu Arg 115 120 125 GlnIle Val Phe Arg Thr Ile Glu Ser Leu Pro Glu Asp Leu Arg Met 130 135 140Ala Ile Thr Leu Arg Glu Leu Asp Gly Leu Ser Tyr Glu Glu Ile Ala 145 150155 160 Ala Ile Met Asp Cys Pro Val Gly Thr Val Arg Ser Arg Ile Phe Arg165 170 175 Ala Arg Glu Ala Ile Asp Asn Lys Val Gln Pro Leu Ile Arg Arg180 185 190 6 144 PRT Haemophilus influenzae 6 Phe Leu Ser Ala Phe LysAsn Leu Ala Asn Phe Lys Arg Gln Ser Ala 1 5 10 15 Phe Lys Thr Trp IlePhe Ala Ile Leu Lys Asn Lys Ile Ile Asp Tyr 20 25 30 Leu Arg Gln Lys GlyArg Phe Val Leu Glu Ser Glu Leu Glu Asp Glu 35 40 45 Asn Thr Asn Asn SerPhe Phe Asp Glu Lys Gly His Trp Lys Pro Glu 50 55 60 Tyr His Pro Ser GluLeu Gln Gly Glu Glu Glu Thr Val Tyr Ser Asp 65 70 75 80 Glu Phe Trp LeuIle Phe Glu Thr Cys Leu Asn Cys Leu Pro Ala Lys 85 90 95 Gln Ala Lys IlePhe Met Met Arg Glu Phe Leu Glu Leu Ser Ser Glu 100 105 110 Glu Ile CysGln Glu Thr His Leu Thr Ser Ser Asn Leu His Thr Thr 115 120 125 Leu TyrArg Ala Arg Leu Gln Leu Gln Asn Cys Leu Ser Lys Lys Leu 130 135 140 7 30DNA Mycobacteria leprae 7 atgaacgaac tgctcgagat cttgcctgcc 30 8 30 DNAMycobacteria leprae 8 tcacccgccg cgacgatctc ggacgtcaac 30 9 33 DNAArtificial Sequence Sigma promoter consensus sequence 9 gaacttnnnnnnnnnnnnnn nnntctgann nnn 33

What is claimed is:
 1. An isolated, purified DNA, wherein said DNA has asequence selected from the group consisting of the sequence shown inSEQ. ID. NO. 1 and sequence conservative variants thereof.
 2. A DNAvector comprising the DNA of claim 1 operably linked to a transcriptionregulatory element.
 3. A cell comprising a DNA vector as defined inclaim 2, wherein said cell is selected from the group consisting ofbacterial, fungal, plant, insect, and mammalian cells.
 4. A cell asdefined in claim 3, wherein said cell is bacterial cell.